Alcohol dehydrogenase gene of acetic acid bacterium

ABSTRACT

A novel gene having a function of improving acetic acid fermentation in practical level was cloned from a practical acetic acid bacterium belonging to the genus  Gluconacetobacter  by a method for obtaining a gene having a growth-promoting function in acetic acid-containing medium from the chromosomal DNA library of the acetic acid bacterium. It was made possible to significantly shorten the growth induction period and significantly improve the acetic acid fermentation rate of transformants obtained by introducing the gene into acetic acid bacteria, when such transformants are cultured in the presence of ethanol.

FIELD OF THE INVENTION

The present invention relates to a gene encoding a protein having agrowth-promoting function derived from a microorganism, a microorganismwherein the number of copies of such gene is amplified, particularlyacetic acid bacteria belonging to the genus Acetobacter or the samebelonging to the genus Gluconacetobacter, and a method for efficientlyproducing vinegar containing acetic acid at a high concentration usingthese microorganisms.

BACKGROUND OF THE INVENTION

Acetic acid bacteria are microorganisms broadly utilized for vinegarproduction. In particular, acetic acid bacteria belonging to the genusAcetobacter or the same belonging to the genus Gluconacetobacter areutilized for industrial acetic acid fermentation.

In acetic acid fermentation, ethanol in media is oxidized and convertedinto acetic acid by acetic acid bacteria, and as a result, acetic acidis accumulated in the media. However, acetic acid has an inhibitoryaction on acetic acid bacteria. As the amount of accumulated acetic acidincreases and the acetic acid concentration in media becomes higher, thegrowth ability and the fermentation ability of acetic acid bacteriagradually decrease.

In particular, growth induction period, that is, the period until aceticacid bacteria actually start to grow, and then it becomes possible toconfirm the accumulation of acetic acid, tends to be longer as aceticacid concentration becomes higher.

Hence, in acetic acid fermentation, it is desired to further shorten thegrowth induction period, even in the case of a high acetic acidconcentration. As a means for this purpose, a method has been reportedthat involves adding PQQ (4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f]quinoline-2,7,9-tricarboxylic acid) to a fermentation liquid to promotegrowth, so as to shorten so-called the growth induction period (e.g.,see Patent document 1).

However, obtainment of PQQ in large quantities is difficult and PQQ isexpensive. Thus, implementation of such a method at industrial scale isalways associated with problems. Accordingly, it has been attempted tobreed and improve acetic acid bacteria by promoting the growth of aceticacid bacteria, cloning genes (growth-promoting genes) encoding proteinshaving a function capable of shortening so-called the growth inductionperiod, and using the growth-promoting genes.

However, no genes involved in growth promotion of acetic acid bacteriahave been isolated so far. Under such circumstances, isolation of anovel growth-promoting gene encoding a protein having a function ofpromoting the growth of acetic acid bacteria at practical level and ofshortening the growth induction period and breeding of acetic acidbacteria having a stronger growth function using the growth-promotinggene have been desired.

-   Patent document 1: JP Patent Publication (Kokai) No. 61-58584 A    (1986)-   Patent document 2: JP Patent Publication (Kokai) No. 60-9488 A    (1985)-   Patent document 3: JP Patent Publication (Kokai) No. 60-9489 A    (1985)-   Patent document 4: Specification of JP Patent Application No.    2003-350265-   Non-patent document 1: “Biochimica et Biophysica Acta,” vol. 1088,    pp. 292-300, 1991-   Non-patent document 2: “Applied and Environmental Microbiology,”    vol. 55, pp. 171-176, 1989-   Non-patent document 3: “Agricultural and Biological Chemistry,” vol.    52, pp. 3125-3129, 1988-   Non-patent document 4: “Agricultural and Biological Chemistry,” vol.    49, pp. 2091-2097, 1985-   Non-patent document 5: “Bioscience, Biotechnology and Biochemistry,”    vol. 58, pp. 974-975, 1994-   Non-patent document 6: “European Journal of Biochemistry,” vol. 254,    pp. 356-362, 1998-   Non-patent document 7: “Methods in Enzymology,” vol. 89, pp.    450-457, 1982-   Non-patent document 8: “Methods in Enzymology,” vol. 89, pp.    491-496, 1982-   Non-patent document 9: “Cellulose,” pp. 153-158, 1989

SUMMARY OF THE INVENTION

As described above, no successful relevant cases have been reported,such as elucidation of the growth-promoting function of acetic acidbacteria at the gene level or development of practical acetic acidbacteria having a high growth-promoting function. Hence, the objects ofthe present invention are to isolate a novel alcohol dehydrogenase geneencoding a protein capable of improving the growth-promoting function atpractical level, breed an acetic acid bacterium having a bettergrowth-promoting function using the alcohol dehydrogenase gene, andprovide a method for efficiently producing vinegar with a higher aceticacid concentration using the acetic acid bacterium.

We generated a hypothesis that a specific growth-promoting gene (that isabsent in other microorganisms) is present in acetic acid bacteria,which are capable of growing and fermenting even in the presence ofacetic acid. We then conceived the novel idea that the use of such agene would enable further improvement in the growth-promoting functionof microorganisms than ever before, and would enable development of amethod for producing vinegar containing acetic acid at a highconcentration more efficiently than ever before. Thus, we have completedthe present invention.

The present invention is as follows.

-   (1) A protein ADH, which is either of the following (A) or (B):

(A) a protein ADH, which has the amino acid sequence represented by SEQID NO: 2; or

(B) a protein ADH, which consists of an amino acid sequence derived fromthe amino acid sequence represented by SEQ ID NO: 2 by substitution,deletion, insertion, addition, or inversion of 1 or several amino acidsand has alcohol dehydrogenase activity.

-   (2) The DNA of a gene, which encodes either of the following protein    ADH (A) or (B):

(A) a protein ADH, which has the amino acid sequence represented by SEQID NO: 2; or

(B) a protein ADH, which consists of an amino acid sequence derived fromthe amino acid sequence represented by SEQ ID NO: 2 by substitution,deletion, insertion, addition, or inversion of 1 or several amino acidsand has alcohol dehydrogenase activity.

-   (3) A gene, which consists of the following DNA (A), (B), or (C):

(A) a DNA, which contains the nucleotide sequence consisting ofnucleotide Nos. 359 to 1390 within the nucleotide sequence representedby SEQ ID NO: 1,

(B) a DNA, which hybridizes under stringent conditions to a DNAconsisting of a nucleotide sequence complementary to the DNA consistingof the nucleotide sequence of nucleotide Nos. 359 to 1390 within thenucleotide sequence represented by SEQ ID NO: 1 and encodes a proteinADH having alcohol dehydrogenase activity; or

(C) a DNA, which hybridizes under stringent conditions to a DNAconsisting of a nucleotide sequence that can be a probe prepared from apart of the DNA consisting of the nucleotide sequence of nucleotide Nos.359 to 1390 within the nucleotide sequence represented by SEQ ID NO: 1and encodes the protein ADH having alcohol dehydrogenase activity.

-   (4) A recombinant vector, which contains the DNA of (2) or (3).-   (5) A transformant, which contains the recombinant vector of (4).-   (6) A microorganism, which has alcohol dehydrogenase activity    enhanced by an amplified number of copies of the DNA of (2) or (3)    within a cell.-   (7) The microorganism of (6), which is an acetic acid bacterium    belonging to the genus Acetobacter or the genus Gluconacetobacter.-   (8) A method for producing vinegar, which comprises culturing the    microorganism of (6) or (7) in a medium containing alcohol and    causing the microorganism to produce and accumulate acetic acid in    the medium.-   (9) Vinegar containing acetic acid at a high concentration, which is    obtained by the method of (8).-   (10) The vinegar of(9), wherein the acetic acid concentration ranges    from 10% to 13%.

According to the present invention, it is possible to impart agrowth-promoting function to microorganisms and enhance the function.Furthermore, to microorganisms having ability to oxidize alcohol,particularly to acetic acid bacteria, ability to significantly shortenthe growth induction period and efficiently accumulate acetic acid at ahigh concentration in a medium can be imparted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the restriction enzyme map of the genefragment (pP1) derived from Gluconacetobacter entanii.

FIG. 2 shows the fermentation process of the transformant that has anamplified number of copies of the NAD-dependent alcohol dehydrogenasegene derived from Gluconacetobacter entanii.

FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) of a protein encodedby the NAD-dependent alcohol dehydrogenase gene derived fromGluconacetobacter entanii.

FIG. 4 shows the nucleotide sequence of primer 1 (SEQ ID NO: 3).

FIG. 5 shows the nucleotide sequence of primer 2 (SEQ ID NO: 4).

The present invention is explained in detail as follows. Thisapplication claims a priority of Japanese patent application No.2003-66697 filed on Mar. 12, 2003, and encompasses the content describedin the specification and/or drawings of the Japanese patent application.

As a result of intensive studies concerning a method for discovering agrowth-promoting gene from acetic acid bacteria, we have developed amethod for isolating a growth-promoting gene from acetic acid bacteriaby constructing a chromosomal DNA library of an acetic acid bacterium,transforming the chromosomal DNA library into acetic acid bacteria, andthen screening for a gene that enables a strain to grow within 3 days onagar media in the presence of 1% acetic acid, whereas the straingenerally requires 4 days to grow on the same media.

By the use of this method, we have succeeded for the first time incloning a novel alcohol dehydrogenase gene (hereinafter it may also bereferred to as the adh gene) capable of improving the growth-promotingfunction at a practical level from acetic acid bacteria belonging to thegenus Gluconacetobacter, which are actually used for vinegar production.

The thus obtained adh gene has homology to some extent with a group ofproteins produced by an adhP gene that has been discovered inEscherichia coli, an adh A gene in Shinorhizobium meliloti and the like,as a result of homology search of DDBJ/EMBL/Genbank and SWISS-PROT/PIR.It was inferred that the adh gene is an alcohol dehydrogenase gene (adhgene) of acetic acid bacteria.

However, the adh gene of the present invention has extremely lowhomology of 6.4% at the amino acid sequence level with an adh gene(e.g., see Non-patent document 1) of an acetic acid bacterium(Acetobacter polyoxogenes) that has been reported so far. Moreover, thepredicted molecular weight of the adh gene of the present inventiondiffers greatly from that of the adh gene as reported in Non-patentdocument 1. Hence, it has been confirmed that the adh gene of thepresent invention clearly differs from that of any adh gene as amembrane protein that has been reported to date.

Furthermore, the adh gene of the present invention has 39% homology atthe amino acid sequence level with the adhP gene of Escherichia coli andhas 56% homology at the amino acid sequence level with the adhA gene ofShinorhizobium meliloti. Because of such extremely low degrees ofhomology, it was confirmed that the adh gene of the present invention issomewhat similar to adh genes of other prokaryotes, but is a novel geneencoding a novel protein (also referred to as protein ADH) specific toacetic acid bacteria.

In the present invention, a transformant having an amplified number ofcopies of the adh gene was prepared by ligating the adh gene to aplasmid vector and then transforming an acetic acid bacterium with thevector. It was confirmed that the gene encodes a protein having alcoholdehydrogenase activity of the acetic acid bacterium, because alcoholdehydrogenase activity is enhanced by approximately 54 times in thetransformant.

We have also discovered that when aeration culture is carried out in thepresence of ethanol, acetic acid fermentation ability, particularly agrowth-promoting function, is significantly improved, and the growthinduction period is shortened. Therefore, vinegar with a high aceticacid concentration can be efficiently produced. Thus, we have completedthe present invention.

The present invention will be described in detail as follows.

(1) DNA of the present invention

The DNA of the present invention has homology to some extent with an adhgene of Escherichia coli or the like and contains a nucleotide sequencethat can encode alcohol dehydrogenase having the amino acid sequencerepresented by SEQ ID NO: 2 in the sequence listing, which can improvethe growth-promoting function. The DNA of the present invention containsregulatory element and structural gene.

The DNA of the present invention can be obtained from the chromosomalDNA of Gluconacetobacter entanii as described below.

First, a chromosomal DNA library of Gluconacetobacter entanii, such asthe Acetobacter altoacetigenes MH-24 strain (deposited under accessionnumber FERM BP-491 on Feb. 23, 1984, (original deposition) with theInternational Patent Organism Depositary (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan), the National Institute of AdvancedIndustrial Science and Technology (AIST)), is prepared. In addition, thechromosomal DNA is obtained by a conventional method (e.g., see Patentdocument 3).

Next, to isolate an alcohol dehydrogenase gene from the obtainedchromosomal DNA, a chromosomal DNA library is constructed. First, thechromosomal DNA is partially digested with appropriate restrictionenzymes to obtain a mixture of various fragments. Through the regulationof time for cleavage reaction and the like so as to regulate the degreesof cleavage, wide-ranging types of restriction enzymes can be used. Forexample, the chromosomal DNA is digested by causing Satu3A I to act onthe DNA at 30° C. or more, preferably at 37° C., at enzyme concentrationranging from 1 to 10 units/ml for various reaction time (1 minute to 2hours). In addition, Pst I was used in Examples shown below.

Next, the thus cleaved chromosomal DNA fragment is ligated to a vectorDNA that is autonomously replicable within acetic acid bacteria, therebyconstructing a recombinant vector. Specifically, a restriction enzyme(such as Pst I, which causes the generation of a terminal nucleotidesequence complementary to the restriction enzyme Pst I used for thecleavage of the chromosomal DNA) is caused to act on the vector DNAunder conditions of 37° C. and enzyme concentration ranging from 1 to100 units/ml for 1 or more hours, thereby completely digesting andcleaving the vector DNA.

Next, the above-obtained mixture of chromosomal DNA fragments is mixedwith the cleaved vector DNA, and then T₄ DNA ligase is caused to actthereon under conditions in which temperature ranges from 4° C. to 16°C. and enzyme concentration ranges from 1 to 100 units/ml for 1 or morehours (preferably 6 to 24 hours), thereby obtaining a recombinantvector.

An acetic acid bacterium that generally requires 4 days to grow in thepresence of 1% acetic acid concentration on agar medium, such as theAcetobacter aceti No. 1023 strain (deposited under accession number FERMBP-2287 on Jun. 27, 1983, (original deposition) with the NationalInstitute of Advanced Industrial Science and Technology, theInternational Patent Organism Depositary (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan)) is transformed using the thusobtained recombinant vector. Subsequently, the resultant is spread onagar medium containing 1% acetic acid, followed by 3 days of culture.The generated colonies are inoculated and cultured in liquid medium.Plasmids are collected from the thus obtained bacterial cells, so thatDNA fragments containing the adh gene can be obtained.

A specific example of the DNA of the present invention the nucleotidesequence of SEQ ID NO: 1 in the sequence listing. In such DNA, anucleotide sequence consisting of nucleotide Nos. 359 to 1390 is acoding region.

The nucleotide sequence represented by SEQ ID NO: 1 in the sequencelisting or the amino acid sequence represented by SEQ ID NO: 2(corresponding to FIG. 3: nucleotide Nos. 359 to 1390) in the sequencelisting showed 39% homology at the amino acid sequence level with theadhP gene of Escherichia coli and showed 56% homology at the amino acidsequence level with the adhA gene of Shinorhizobium moliloti as a resultof homology search of DDBJ/EMBL/Genbank and SWISS-PROT/PIR. Thus, it wasinferred that the relevant gene encodes a protein ADH. However, becauseboth homologies were as low as 60% or less, it was clear that the geneis a novel gene differing from these genes.

Furthermore, obtainment of an adh gene from acetic acid bacterium,Acetobacter polyoxogenes, has been reported (e.g., see Non-patentdocument 1). Amino acid sequence homology between the protein encoded bythe adh gene in this case and the protein encoded by the nucleotidesequence consisting of nucleotide Nos. 359 to 1390 is as very low as6.4%. The gene of the present invention clearly differs from known adhgenes and is clearly a novel adh gene of acetic acid bacteria.

The nucleotide sequence of the DNA of the present invention has beenclarified. Thus, the DNA can be obtained by, for example, polymerasechain reaction (PCR reaction) using genome DNA of an acetic acidbacterium, Gluconacetobacter entanii, as a template, andoligonucleotides synthesized based on the nucleotide sequence, asprimers, or by hybridization using an oligonucleotide synthesized basedon the nucleotide sequence as a probe.

Oligonucleotides can be synthesized according to a conventional methodusing, for example, various commercially available DNA synthesizers.Furthermore, PCR reaction can be carried out according to a conventionalmethod using the Thermal Cycler Gene Amp PCR system 9700 produced byApplied Biosystems, Taq DNA polymerase (produced by TAKARA BIO INC.),KOD-Plus- (produced by TOYOBO CO., LTD.), and the like.

The DNA of the present invention encoding the protein ADH having agrowth-promoting function may be any DNA that encodes a protein obtainedby deletion, substitution, insertion, or addition or inversion of 1 orseveral amino acids at 1 or a plurality of positions, as long as thealcohol dehydrogenase activity or the growth-promoting function of theprotein ADH to be encoded is not deteriorated.

Such a DNA encoding a protein ADH substantially identical to alcoholdehydrogenase having a growth-promoting function can be obtained alsoby, for example, site-directed mutagenesis, specifically by altering thenucleotide sequence by deletion, substitution, insertion, or addition orinversion of amino acids at specific sites. Moreover, the above-alteredDNA can also be obtained by a conventionally known treatment to causemutation.

Furthermore, it is generally known that the amino acid sequence of aprotein and the nucleotide sequence encoding the protein differ slightlybetween species, strains, variants, and varieties. DNAs encodingsubstantially identical proteins can be obtained from overall aceticacid bacteria, particularly those of species, strains, variants, andvarieties of the genus Acetobacter or the genus Gluconacetobacter.

Specifically, a DNA that hybridizes under stringent conditions to, forexample, a DNA consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of nucleotide Nos. 359 to 1390 (of thenucleotide sequence of SEQ ID NO: 1 in the sequence listing) or a DNAconsisting of a nucleotide sequence that can be a probe prepared from apart of the nucleotide sequence consisting of nucleotide Nos. 359 to1390 (of the nucleotide sequence of SEQ ID NO: 1 in the sequencelisting); and encodes a protein having a function to enhance resistanceto acetic acid is isolated from acetic acid bacteria of the genusAcetobacter or the genus Gluconacetobacter, mutated acetic acid bacteriaof the genus Acetobacter or the genus Gluconacetobacter, or naturallymutated strains or varieties thereof In this way, a DNA encoding aprotein substantially identical to the protein can also be obtained. Theterm “stringent conditions” used herein means conditions wherebyso-called specific hybrids are formed and non-specific hybrids are notformed. It is difficult to precisely represent such conditions innumerical values. Examples of such conditions include conditions whereinnucleotide sequences sharing high homology, for example, DNAs sharing70% or more homology, hybridize to each other and nucleotide sequencessharing homology lower than this level do not hybridize to each other.Other such examples include general washing conditions forhybridization, such as conditions wherein washing is carried out at 60°C. with a salt concentration corresponding to 0.1% SDS in the case of1×SSC.

(2) Acetic acid bacteria in the present invention Acetic acid bacteriain the present invention are belonging to the genus Acetobacter or tothe genus Gluconacetobacter, both of which have enhanced resistance toacetic acid.

Specific examples of bacteria of the genus Acetobacter includeAcetobacter aceti such as the Acetobacter aceti No. 1023 strain(deposited with the International Patent Organism Depositary under FERMBP-2287), the Acetobacter aceti subsp. xylinum IF03288 strain, and theAcetobacter aceti IF03283 strain.

Furthermore, examples of bacteria of the genus Gluconacetobacter includethe Gluconacetobacter europaeus DSM6160 strain and Gluconacetobacterentanii such as Acetobacter altoacetigenes. MH-24 strain that iscurrently deposited with the International Patent Organism Depositaryunder FERM BP-491.

The growth-promoting function is enhanced by, for example, amplifyingthe number of copies of the adh gene within the cells or transformingbacteria of the genus Acetobacter using recombinant DNA obtained byligating a DNA fragment containing the structural gene to a promotersequence that efficiently functions in bacteria of the genusAcetobacter.

The growth-promoting function can also be enhanced by substituting apromoter sequence of the gene on a chromosomal DNA with another promotersequence that efficiently functions in acetic acid bacteria of the genusAcetobacter or the genus Gluconacetobacter. Examples include a promotersequence derived from a microorganism other than acetic acid bacteria,such as a promoter of each gene of an ampicillin resistance gene of aplasmid pBR322 (produced by TAKARA BIO INC.) of Escherichia coli, akanamycin resistance gene of a plasmid pHSG298 (produced by TAKARA BIOINC.), a chloramphenicol resistance gene of a plasmid pHSG396 (producedby TAKARA BIO INC.), and a β-galactosidase gene.

The number of copies of the gene within the cells can be amplified byintroducing a multi-copy vector retaining the gene into cells of aceticacid bacteria of the genus Acetobacter. Specifically, amplification canalso be carried out by introducing a plasmid retaining the gene, atransposon retaining the gene, or the like into cells of acetic acidbacteria of the genus Acetobacter or the genus Gluconacetobacter.

Examples of a multi-copy vector (acetic acid bacterium-Escherichia colishuttle vector) include pMV24 (e.g., see Non-patent document 2), pGI18(e.g., see Patent document 4), pUF106 (e.g., see Non-patent document 9),pTA5001 (A), and pTA5001 (B) (e.g., see Patent document 2). Anotherexample of this is pMVLI (e.g., see Non-patent document 3), which is achromosomal integrating vector. Furthermore, examples of a transposoninclude Mu and IS1452.

DNA can be introduced into acetic acid bacteria of the genus Acetobacteror the genus Gluconacetobacter by a calcium chloride method (e.g., seeNon-patent document 4), an electroporation method (e.g., see Non-patentdocument 5), or the like.

A transformant is obtained by introducing a recombinant plasmid intoAcetobacter aceti No. 1023 (FERM BP-2287). Such a recombinant plasmidcomprises at least a DNA fragment having the nucleotide sequencerepresented by SEQ ID NO: 1 in the sequence listing, such as arecombinant plasmid pADH1 prepared by inserting the above DNA fragmentinto an acetic acid bacterium-Escherichia coli shuttle vector(multi-copy vector) pMV24.

When alcohol dehydrogenase activity is enhanced as described above inacetic acid bacteria of the genus Acetobacter or the genusGluconacetobacter having ability to oxidize alcohol, thegrowth-promoting function is enhanced and the growth induction period isshortened, so that vinegar containing acetic acid at a highconcentration can be efficiently produced.

(3) Method for producing vinegar Acetic acid bacteria of the genusAcetobacter or the genus Gluconacetobacter having a selectively enhancedgrowth-promoting function are obtained through the above-describedamplification of the number of copies of the adh gene. Such acetic acidbacteria having ability to oxidize alcohol are cultured in a mediumcontaining alcohol and then caused to produce and accumulate acetic acidin the medium, so that vinegar containing acetic acid at a highconcentration can be efficiently produced.

Acetic acid fermentation in the production method of the presentinvention may be carried out in a manner similar to that used in aconventional vinegar production by fermentation using acetic acidbacteria. A medium to be used for acetic acid fermentation may be eithera synthetic or a natural medium as long as it contains carbon sources,nitrogen sources, inorganic substances, and ethanol, and, if necessary,contains appropriate amounts of nutrition sources required for thegrowth of the employed microbial strain.

Examples of carbon sources include various carbohydrates such asglucose, sucrose and various organic acids. As nitrogen sources, naturalnitrogen sources such as peptone and decomposed products of thefermentation microorganisms can be used.

Furthermore, culture is carried out under aerobic conditions such asthose of a static culture method, a shaking culture method, and anaeration and agitation culture method. Culture is carried out at atemperature ranging from 20° C. to 40° C., preferably 25° C. to 35° C.,and generally at 30° C. pH of a medium generally ranges from 2.5 to 7and preferably ranges from 2.7 to 6.5. pH can also be adjusted usingvarious acids, various nucleotides, buffers, and the like. Generally by1 to 21 days of culture, acetic acid is accumulated at a highconcentration in the medium. The acetic acid concentration of vinegarachieved by the use of this method ranges from 10% to 13%.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be further described specifically byreferring to examples.

EXAMPLE 1 Cloning of a Gene Having Growth-Promoting Function fromGluconacetobacter entanii and Determination of the Nucleotide Sequenceand the Amino Acid Sequence thereof

(1) Construction of Chromosomal DNA Library

The Acetobacter altoacetigenes MH-24 strain (FERM BP-49 1), which is astrain of Gluconacetobacter entanii, was cultivated in shaking culturein YPG medium (3% glucose, 0.5% yeast extract, and 0.2% polypeptone)supplemented with 6% acetic acid and 4% ethanol at 30° C. After thecultivation, the culture medium was centrifuged (7,500×g for 10minutes), thereby obtaining bacterial cells. From the thus obtainedbacterial cells, chromosomal DNAs were prepared by a chromosomal DNApreparation method (e.g., see Patent document 3).

The above-obtained chromosomal DNAs were partially digested with arestriction enzyme Pst I (produced by TAKARA BIO INC.). Escherichiacoi-acetic acid bacterium shuttle vector pMV24 was completely digestedwith the restriction enzyme Pst I. Appropriate amounts of these DNAswere mixed in and then ligated using a ligation kit (TaKaRa DNA LigationKit Ver. 2, produced by TAKARA BIO INC.), thereby constructing achromosomal DNA library of Gluconacetobacter entanii.

(2) Cloning of a gene having growth-promoting function

The chromosomal DNA library of Gluconacetobacter entanii obtained asdescribed above was transformed into Acetobacter aceti No. 1023 strainthat generally requires 4 days to grow on agar medium containing 1%acetic acid, and then cultured on YPG agar medium containing 1% aceticacid and 100 μg/ml ampicillin at 30° C. for 3 days. Colonies generatedwithin 3 days were inoculated and cultured on YPG medium containing 100μg/ml ampicillin and then plasmids were collected from the obtainedbacterial cells. An approximately 1.7-kbp Pst I fragment was cloned asshown in FIG. 1, and the plasmid was named pP1. Furthermore, it could beconfirmed that a fragment that had enabled the Acetobacter aceti No.1023 strain to grow within 3 days on the YPG agar medium containing 1%acetic acid was an approximately 1.2-kbp Bgl I fragment in theapproximately 1.7-kbp Pst I fragment cloned into pP1.

As described above, a gene fragment was obtained that enables theAcetobacter aceti No. 1023 strain to grow within 3 days on agar mediumcontaining. 1% acetic acid, although the strain generally requires 4days to grow on such agar medium containing 1% acetic acid.

(3) Determination of the Nucleotide Sequence of the Cloned DNA Fragment

The above cloned Pst I fragment was inserted into the Pst I site ofpUC19, and then the nucleotide sequence of the fragment was determinedby Sanger's dideoxy chain termination method. As a result, thenucleotide sequence of SEQ ID NO: 1 was determined. Sequencing wascarried out for the entire region of both DNA strands with all thecleavage points overlapping with each other.

In the nucleotide sequence of SEQ ID NO: 1, the presence of an openreading frame encoding 344 amino acids as described in SEQ ID NO: 2(FIG. 3) ranging from nucleotide No. 359 to nucleotide No. 1390 wasconfirmed.

EXAMPLE 2 Effect of Shortening the Growth Induction Period in aTransformant with a Gene Having Growth-Promoting Function Derived fromGluconacetobacter entanii

(1) Transformation into Acetobacter aceti

The above-cloned gene having the growth-promoting function derived fromthe Acetobacter altoacetigenes MH-24 strain (FERM BP-491) was amplifiedby the PCR method using KOD-Plus- (produced by TOYOBO CO., LTD.). Thethus amplified DNA fragment was inserted into the restriction enzyme SmaI cleavage site of the acetic acid bacterium-Escherichia coli shuttlevector pMV24 (e.g., see Non-patent document 2), so as to prepare aplasmid pADH1. The amplified fragment inserted in pADH1 is schematicallyshown in FIG. 1. FIG. 1 shows the restriction enzyme map of theGluconacetobacter entanii-derived gene fragment (pP1) cloned using PstI, the position of the gene having the growth-promoting function, andthe fragment inserted into pADH1.

The PCR method was carried out as described in detail below.Specifically, PCR was carried out under the following PCR conditionsusing a genome DNA of the Acetobacter altoacetigenes MH-24 strain as atemplate, primer 1 (the nucleotide sequence thereof is shown in SEQ IDNO: 3 (FIG. 4)), primer 2 (the nucleotide sequence thereof is shown inSEQ ID NO: 4 (FIG. 5)), and KOD-Plus- (produced by TOYOBO CO., LTD.).

Specifically, the PCR method was carried out for 30 cycles, each ofwhich consisted of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68°C. for 1 minute.

The pADH1 was transformed into the Acetobacter aceti No. 1023 strain byan electroporation method (e.g., see Non-patent document 5). Thetransformant was selected using YPG agar medium supplemented with 100μg/ml ampicillin and 1% acetic acid.

Plasmids were extracted from the ampicillin-resistant transformant thathad grown on the selection medium within 3 days and then analyzedaccording to a conventional method. Thus, it was confirmed that thestrain retained plasmids having the gene with the growth-promotingfunction.

(2) Growth-Promoting Function of the Transformant

The above-obtained ampicillin-resistant transformant having the plasmidpADH1 and the original Acetobacter aceti No. 1023 strain having only theshuttle vector pMV24 introduced therein were compared in terms of growthin YPG medium supplemented with acetic acid.

Specifically, shaking culture (150 rpm) was carried out at 30° C. in 100ml of YPG medium containing 3% acetic acid, 3% ethanol, and 100 μg/mlampicillin. The transformant and the original strain were compared interms of growth in the medium supplemented with acetic acid by measuringabsorbance at 660 nm.

As a result, as shown in FIG. 2, it could be confirmed that thetransformant grew faster than the original Acetobacter aceti No. 1023strain in the medium supplemented with 3% acetic acid and 3% ethanol.The effect of enhancing the growth-promoting function of the gene couldbe confirmed.

(3) Various Enzyme Activities of the Transformant and the OriginalStrain

For the ampicillin-resistant transformant having the plasmid pADH1 andthe original Acetobacter aceti No. 1023 strain having only the shuttlevector pMV24 introduced therein, NAD-dependent alcohol dehydrogenaseactivity was measured with the modified method used in bacteria (e.g.,see Non-patent document 6). Specifically, an appropriate amount ofdisrupted cell suspension was added to 1 ml of reaction mixturecontaining 25 mM sodium phosphate buffer (pH 7.5), 500 mM ethanol, and1.5 mM NAD+, and then absorbance at 340 nm was measured at 25° C. Theamount of 1 μmol of NADH produced per minute was determined tocorrespond to 1U of activity. Furthermore, membrane-bound alcoholdehydrogenase activity was measured with the method used in acetic acidbacteria (e.g., see Non-patent document 7) and membrane-bound aldehydedehydrogenase activity was measured with the method used in acetic acidbacteria (e.g., see Non-patent document 8). The results are shown inTable 1.

TABLE 1 NAD-dependent Membrane-bound Membrane-bound alcohol alcoholaldehyde dehydrogenase dehydrogenase dehydrogenase (U/mg) (U/mg) (U/mg)Original 0.17 2.17 8.63 strain Transformant 9.19 1.82 7.01

Based on the results in Table 1, the transformant was found to havemembrane-bound alcohol dehydrogenase activity and aldehyde dehydrogenaseactivity at the same levels as those of the original Acetobacter acetiNo. 1023 strain. However, the transformant had NAD-dependent alcoholdehydrogenase activity that was higher by approximately 54 times thanthat of the original strain. Thus, it was confirmed that the cloned genewas the adh gene encoding NAD-dependent alcohol dehydrogenase.

EXAMPLE 3 Acetic Acid Fermentation Test for the Transformant with theAdh Gene Derived from Gluconacetobacter entanii

The ampicillin-resistant transformant having the plasmid pADH1 obtainedin Example 2 and the original Acetobacter aceti No. 1023 strain havingonly the shuttle vector pMV24 were compared in terms of acetic acidfermentation ability.

Specifically, aeration and agitation culture (30° C., 400 rpm, and 0.20vvm) was carried out using a 51 mini-jar fermentor (produced by MitsuwaScientific Corp.; KMJ-5A) containing 2.51 of YPG medium containing 1%acetic acid, 4% ethanol, and 100 μg/ml ampicillin. The strains werecaused to ferment so as to result in an acetic acid concentration of 3%.Subsequently, each culture medium was removed while leaving 700 ml ofthe culture medium in the mini-jar fermentor. To the remaining 700 ml ofthe culture medium, 1.81 of YPG medium containing acetic acid, ethanol,and 100 μg/ml ampicillin was added. The medium was adjusted to result in3% and 4% concentrations of acetic acid and ethanol, respectively, andthen acetic acid fermentation was initiated again. Aeration andagitation culture was continued while adding ethanol to maintain 1%ethanol concentration in the media during fermentation. The transformantand the original strain were compared in terms of acetic acidfermentation ability. The results are summarized in Table 2.

TABLE 2 Final acetic Growth acid Specific Production inductionconcentration growth rate rate period achieved (%) (OD660/hr) (%/hr)(hr) Original 9.9 0.0162 0.071 54.4 strain Transformant 10.5 0.01160.089 5.0

Based on the results in Table 2, the growth induction period wassignificantly shortened in the transformant compared with the case ofthe original strain. Thus, it could be confirmed that the transformantwas able to carry out acetic acid fermentation efficiently.

EXAMPLE 4 Acetic Acid Fermentation Test for the Transformant with theAdh Gene Derived from Gluconacetobacter entanii

(1) Transformation into Acetobacter altoacetigenes

The gene having the growth-promoting function derived from theAcetobacter altoacetigenes MH-24 strain (FERM BP-491) was amplified bythe PCR method using KOD-Plus- (produced by TOYOBO CO., LTD.). Aftercleavage of the acetic acid bacterium-Escherichia coli shuttle vectorpGI18 (e.g., see Patent document 4) with the restriction enzyme Smia I,the thus amplified DNA fragment was inserted into the site, so as toprepare a plasmid pADH2. The amplified fragment inserted into pADH2 isschematically shown in FIG. 1. FIG. 1 shows the restriction enzyme mapof the Gluconacetobacter entanii-derived gene fragment (pP1) clonedusing Pst I, the position of the gene having the growth-promotingfunction, and the fragment inserted into pADH2.

The PCR method was carried out as described in detail below.Specifically, PCR was carried out under the following PCR conditionsusing a genome DNA of the Acetobacter altoacetigenes MH-24 strain as atemplate, primer 1 (the nucleotide sequence thereof is shown in SEQ IDNO: 3 (FIG. 4)), and primer 2 (the nucleotide sequence thereof is shownin SEQ ID NO: 4 (FIG. 5)), and KOD-Plus- (produced by TOYOBO CO., LTD.).

Specifically, the PCR method was carried out for 30 cycles, each ofwhich consisted of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68°C. for 1 minute.

The pADH2 was transformed into the Acetobacter altoacetigenes MH-24strain by the electroporation method (e.g., see Non-patent document 5).The transformant was selected using YPG agar medium supplemented with100 μg/ml ampicillin, 4% acetic acid, and 3% ethanol.

Plasmids were extracted from the ampicillin-resistant transformant thathad grown on the selection medium and then analyzed according to aconventional method. Thus, it was confirmed that the transformantretained plasmids having the adh gene.

(2) Acetic acid fermentation test for the transformant

The ampicillin-resistant transformant having the plasmid pADH2 obtainedin

(1) and the original Acetobacter altoacetigenes MH-24 strain having onlythe shuttle vector pGI18 introduced therein were compared in terms ofacetic acid fermentation ability.

Specifically, aeration and agitation culture (30° C., 500 rpm, and 0.20vvm) was carried out using a 5 mini-jar fermentor (produced by MitsuwaScientific Corp.; KMJ-5A) containing 2.5 of raw-material medium (7%acetic acid, 3% ethanol, 0.2% yeast extract, and 0.2% glucose)containing 100 μg/ml ampicillin. At the stage where the growth of thebacteria was clearly observed and the residual ethanol concentrationreached 2%, ethanol-containing medium (1% acetic acid, 50% ethanol, 0.2%yeast extract, and 0.2% glucose) was fed so that the ethanolconcentration of the fermentation liquid was controlled to be 2%. Withthis method for acetic acid fermentation, acetic acid fermentationability was compared between the transformant and the original strain.The results are summarized in Table 3.

TABLE 3 Final acetic acid Specific growth concentration rate Productionrate achieved (%) (OD660/hr) (%/hr) Original strain 15.6 0.0061 0.31Transformant 17.3 0.0045 0.17

Based on the results in Table 3, it could be confirmed that thetransformant was significantly superior to the original strain in termsof the final acetic acid concentration achieved.

EXAMPLE 5 Acetic Acid Fermentation Test for the Transformant with theAdh Gene Derived from Gluconacetobacter entanii

(1) Transformation into Acetobacter aceti subsp. xylinum

The plasmid pADH2 obtained in Example 4 was transformed into theAcetobacter aceti subsp. xylinum IFO3288 strain that is a strain ofAcetobacter aceti subsp. xylinum by the electroporation method (seeNon-patent document 5). The transformant was selected using YPG agarmedium supplemented with 100 μg/ml ampicillin.

Plasmids were extracted from the ampicillin-resistant transformant thathad grown on the selection medium and then analyzed according to aconventional method. Thus, it was confirmed that the strain retainedplasmids having the gene involved in enhanced resistance to acetic acid.

(2) Acetic acid fermentation test

The ampicillin-resistant transformant having the plasmid pADH2 obtainedin (1) and the original Acetobacter aceti subsp. xylinum IFO3288 strainhaving only the shuttle vector pGI18 introduced therein were compared interms of acetic acid fermentation ability.

Specifically, aeration and agitation culture (30° C., 500 rpm, and 0.20vvm) was carried out in a raw-material medium (alcohol concentration of7.8% and acetic acid concentration of 0.26%) that had been prepared bymixing 17.9% saccharified rice solution, 3.2% fermented moromi, 7.8%alcohol, and 71.1% water in a 5 1 mini-jar fermentor (produced byMitsuwa Scientific Corp.; KMJ-5A). Continuous fermentation was carriedout at acetic acid concentration of 7.3%. The strains in continuousfermentation at acetic acid concentration of 7.2% were compared in termsof the raw-material medium addition rate. The results are shown in Table4. Furthermore, the strains were compared in terms of acetic acidfermentation ability, where the raw-material medium addition rate of thetransformant had been adjusted to the raw-material medium addition rateof the original strain in continuous fermentation at acetic acidconcentration of 7.3%. The results are shown in Table 4.

TABLE 4 Raw-material Acetic acid medium addition concentration (%) OD660rate (g/hr) Original strain 7.17 0.538 84.7 Transformant 7.20 0.562 95.5

TABLE 5 Raw-material Acetic acid medium addition concentration (%) OD660rate (g/hr) Original strain 7.31 0.529 87.3 Transformant 7.82 0.466 88.3

Based on the results in Table 4 and Table 5, it could be confirmed thatthe transformant was significantly superior to the original strain interms of continuous acetic acid fermentation, productivity (raw-materialmedium addition rate), and concentration of the produced acetic acid.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, a novel gene involved in acetic acidresistance is provided. Furthermore, a bred strain capable of producingat high efficiency vinegar with a higher acetic acid concentration canbe obtained using the gene. Thus, a method for producing at highefficiency vinegar with a higher acetic acid concentration using thebred strain can be provided.

Sequence Listing Free Text

SEQ ID NO: 3: primer

SEQ ID NO: 4: primer

1. An isolated DNA wherein, said DNA encodes the protein alcoholdehydrogenase (ADH), which has the amino acid sequence of SEQ ID NO: 2.2. An isolated nucleic acid, which comprises the nucleotide sequenceconsisting of nucleotide Nos: 359-1390 of SEQ ID NO:
 1. 3. A recombinantvector, which contains the DNA of claim 1 or
 2. 4. An isolatedtransformed cell, which contains the recombinant vector of claim
 3. 5. Amicroorganism, which has alcohol dehydrogenase activity enhanced by anamplified number of copies of the DNA of claim 1 or 2 within a cell. 6.The microorganism of claim 5, which is an acetic acid bacteriumbelonging to the genus Acetobacter or the genus Gluconacetobacter.
 7. Amethod for producing vinegar, which comprises culturing themicroorganism of claim 5 or 6 in a medium containing alcohol and causingthe microorganism to produce and accumulate acetic acid in the medium.